Relationship Between Antibody-Positive Rate Against Plasmodium Vivax Circumsporozoite Protein and Incidence of Malaria

ISSN (Print) 0023-4001 ISSN (Online) 1738-0006

Korean J Parasitol Vol. 53, No. 2: 169-175, April 2015 ▣ ORIGINAL ARTICLE http://dx.doi.org/10.3347/kjp.2015.53.2.169

Relationship between Antibody-Positive Rate against Plasmodium vivax Circumsporozoite Protein and Incidence of Malaria

Hyeong-Woo Lee1,†, Yoon-Joong Kang2,†, Shin-Hyeong Cho3, Byoung-Kuk Na4, Jhang Ho Pak5, Ho-Woo Nam6, 7 8, 7, Yun-Kyu Park , Youngjoo Sohn *, Tong-Soo Kim * 1Department of Pathology, Immunology, and Laboratory Medicine, College of Medicine, University of Florida, FL 32610, USA; 2Department of Biomedical Science, Jungwon University, Goesan, Chungbuk 367-805, Korea; 3Division of Malaria and Parasitic Diseases, National Institute of Health, Korea Centers for Disease Control and Prevention, Osong 363-951, Korea; 4Department of Parasitology and Tropical Medicine and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751, Korea; 5Asan Institute for Life Sciences, University of Ulsan College of Medicine Asan Medical Center, Seoul 138-736, Korea; 6Department of Parasitology, College of Medicine, Catholic University of Korea, Seoul 137-701, Korea; 7Department of Parasitology and Tropical Medicine and Inha Research Institute for Medical Sciences, Inha University School of Medicine, Incheon 400-712, Korea; 8Department of Anatomy, College of Korean Medicine, Institute of Korean Medicine, Kyung Hee University, Seoul 130-701, Korea

Abstract: The relationship between anti-Plasmodium vivax circumsporozoite protein (CSP) antibody levels and the prevalence of malaria in epidemic areas of South Korea was evaluated. Blood samples were collected from inhabitants of Gim- po-si (city), Paju-si, and Yeoncheon-gun (county) in Gyeonggi-do (province), as well as Cheorwon-gun in Gangwon-do from November to December 2004. Microscopic examinations were used to identify malaria parasites. ELISA was used to quantitate anti-circumsporozoite protein (CSP) antibodies against P. vivax. A total of 1,774 blood samples were collected. The overall CSP-ELISA-positive rate was 7.7% (n= 139). The annual parasite incidences (APIs) in these areas gradu- ally decreased from 2004 to 2005 (1.09 and 0.80, respectively). The positive rate in Gimpo (10.4%, 44/425) was the highest identified by CSP-ELISA. The highest API was found in Yeoncheon, followed by Cheorwon, Paju, and Gimpo in both years. The positive rates of CSP-ELISA were closely related to the APIs in the study areas. These results suggest that se- roepidemiological studies based on CSP may be helpful in estimating the malaria prevalence in certain areas. In addition, this assay can be used to establish and evaluate malaria control and eradication programs in affected areas.

Key words: Plasmodium vivax, annual parasite incidence, seropositivity, circumsporozoite protein

INTRODUCTION peninsula for several centuries prior [2]. As a result of a national malaria eradication program conducted with the in- Plasmodium vivax is a causative agent of relapsing benign ter- volvement of the World Health Organization, the incidence of tian malaria, the second leading type of human malaria, vivax malaria in South Korea has rapidly decreased [3,4]. In which afflicts several hundred millions of individuals annual- fact, vivax malaria was considered eradicated in South Korea ly. The disease is a major public health problem in most tropi- in the late 1970s until 2 sporadic cases were detected in the cal regions, as well as many temperate countries, including 1980s [5]. In 1993, a case was diagnosed among South Korean North and South Korea [1]. The first scientific documentation soldiers serving in Northern Gyeonggi Province [6], and subse- of malaria occurrence in Korea was published in 1913, al- quently, 2 infected civilians were reported [7]. Thereafter, though malaria had been prevalent throughout the Korean many cases have been reported near the demilitarized zone (DMZ), centering on Paju, Yeoncheon, Cheorwon, Gimpo, Received 23 September 2014, revised 2 January 2015, accepted 25 February • Ganghwa, Goyang, and Dongducheon. There is now consider- 2015. † These authors contributed equally to this work. able concern that malaria will become re-established in the re- Corresponding author ([email protected]; [email protected]) * gion and then expand geographically [8]. © 2015, Korean Society for Parasitology and Tropical Medicine The malaria research team at the Korea National Institute of This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) Health (KNIH) has developed several diagnostic methods to which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. support pathological examinations. One of these is an anti-

169 170 Korean J Parasitol Vol. 53, No. 2: 169-175, April 2015

body detection method using circumsporozoite protein (CSP), and parents/kin of child participants in a household provided which is a sporogony antigen and surface membrane protein informed consent. Parents that were unwilling to have their that is expressed in all plasmodial sporozoites. CSP has a cen- children participate were identified, and the data of these chil- tral immunodominant region consisting of a short tandem re- dren were subsequently excluded, without prejudice, from peat of amino acid sequences that contain multiple copies of study surveys. Furthermore, all data were analyzed anony- the immunodominant B-cell epitope [9]. CSP is classified into mously; no patient was identified by the name. Ethical ap- 2 serotypes, which differ with respect to the sequences at the proval of the study was obtained from the KNIH. This study repeated region of the CSP gene: VK210 [the dominant form was conducted according to the principles expressed in the consisting of GDRA(D/A)GQPA repeats] and VK247 [the vari- Declaration of Helsinki. ant form consisting of ANGA(G/D)(N/D)QPG repeats]. It has been established that Korean vivax malaria belongs to the Microscopic examinations VK210 type; thus, in a previous study, Lee et al. [10] construct- Thin blood films were prepared to determine the infectivity ed recombinant CSP from a Korean isolate of the VK210 type. of blood samples. The blood films were fixed with methanol Serological surveys have provided valuable epidemiological and stained with Giemsa solution diluted with buffered water information, especially in areas of low endemicity [11]. The at pH 7.2 to emphasize the parasite inclusions in red blood rate of parasitemia is the foundation of the classical method cells (RBCs). The fixed monolayer of RBCs in this procedure used for measuring the prevalence of malaria. However, the facilitates morphological identification of the parasite to the incidence of parasitemia alone may fail to adequately describe species level and provides greater specificity than that obtained the epidemiology of malaria in a given population. For exam- with thick-film examinations. Thin blood films are often pre- ple, when the incidence of malaria is low, mass blood surveys ferred for routine estimations of parasitemia because the or- do not yield results commensurate with the work involved ganisms are easier to see and count with this method [14]. To [12,13]. In this study, anti-P. vivax CSP antibody levels were estimate the densities of blood-stage parasites by microscopy, determined among populations in epidemic areas, namely, the number of asexual parasites observed relative to 200 white Gimpo, Paju, Yeoncheon, and Cheorwon, to evaluate the effi- blood cells (WBCs) was calculated and then the parasite to cacy of these antigens in determining local malaria prevalence WBC ratio was multiplied by 8,000, which is the assumed compared to the incidence of malaria patients. number of WBCs per microliter of blood [15].

MATERIALS AND METHODS ELISA ELISA was used to determine whether the blood samples Study areas and blood sample collection contained antibodies against the CSP VK210 antigens of P. The study was conducted in Gimpo, Paju, and Yeoncheon vivax [10]. Briefly, the capture antigen solution (50 µl, 0.5 μg/ of Gyeonggi Province and Cheorwon of Gangwon Province, ml) was placed in a 96-well plate (Corning, Lowell, Massachu- South Korea, from November to December of 2004. A blood setts, USA) and incubated for 12 hr at room temperature. The sample was collected from 1,774 individuals and transferred wells were aspirated and filled with blocking buffer which to the KNIH at the Korean Center for Disease Control and Pre- contains 1% bovine serum albumin and 0.05% PBS-Tween vention (KCDC). Blood smears were prepared for microscopic 20, and incubated for 1 hr at room temperature. After the wells examinations. Sera were separated and stored at -20˚C for anti- were washed 3 times with 0.05% PBS-Tween 20, human se- body analysis. Informed consent was obtained from all indi- rum samples in blocking buffer at a dilution of 1:100 (vol/vol) viduals. were added to the wells. Four positive and 4 negative control serum samples were also added to each plate. After 2 hr of in- Ethics statement cubation at room temperature, the plates were washed 3 times All participants were informed of the methodology of the with 0.05% PBS-Tween 20, and then peroxidase-conjugated study and signed an informed consent form according to ethi- anti-human IgG (Sigma; 1:2,000, vol/vol) diluted in blocking cal standards. The study procedures, potential risks, and bene- buffer was added. The plates were re-incubated for 1 hr at fits were explained to all participants. All adult participants room temperature. The reaction was stopped by washing the Lee et al.: Antibody-positive rate of P. vivax CSP and malaria incidence 171

plates as described above. To develop the color, 100 µl of given year. The data were analyzed using SPSS software (ver- 2,2’-azino-di-(3-ethyl-benzthiozoline-6-sulfonic acid) (ABTS) sion 17.0, SPSS Inc.; Chicago, Illinois, USA). A P-value of less peroxidase substrate (Kirkegaard & Perry Laboratories; Gaith- than 0.05 was considered statistically significant. The correla- ersburg, Maryland, USA) was added, and the plates were incu- tion magnitude was interpreted as none (0.0-0.09), small (0.1- bated for 30 min. The absorbance was measured at 405 nm, 0.3), medium (0.3-0.5), or strong (0.5-1.0) [16]. and the cut-off value for positivity was defined as the mean+ 2SD (standard deviation) of the negative control samples. RESULTS

Calculation of the annual parasite incidence (API) The study locations are shown on the map in Fig. 1. All ar- Malaria is classified as a Group III communicable disease eas are near the DMZ, a known high-risk area for malaria. that should be controlled by the Korean Government. Cases of Blood samples were collected from participants residing in 23 malaria detected in private hospitals or clinics are reported to villages in 3 cities (Gimpo, Paju, and Yeoncheon) located in the local Public Health Center (PHC). The data collected by Gyeonggi Province and from 6 villages in Cheorwon in Gang- the PHCs are periodically provided to the Division of Infec- won Province, South Korea. The total number of inhabitants tious Disease Surveillance (DIDS) of the KCDC. Therefore, for in the study areas in 2004 was 92,246; thus, 1.92% of the this study, APIs of 2004 and 2005 were obtained from the whole population was sampled. In Paju, 1 malaria case was DIDS. The API was calculated as the number of malaria-posi- reported in 1993, and subsequently, 3,264 patients were re- tive patients per 1,000 inhabitants for each of the study sites ported up to 2013. In Gimpo, 1 patient was reported in 1995, via microscopy. APIz=(no. of positive slides/total no. of and subsequently, 1,142 patients were reported up to 2013. In slides) ×1,000. Yeoncheon, 2 patients were reported in 1995, and subsequently, 1,935 patients were reported up to 2013. In Cheorwon, 9 Data analysis patients were reported in 1997, and subsequently, 1,388 pa- Pearson’s correlation analysis was performed to examine the tients were reported up to 2013. As shown in Fig. 2, the inci- relationship between seropositivity and the API of P. vivax in a dences of malaria patients reached its lowest levels in 2004,

A B

C D

Fig. 1. Study areas. (A) Gimpo-si, (B) Paju-si, (C) Yeoncheon-gun, and (D) Cheorwon-gun. a, Haseong-myon; b, Wolgot-meon; c, Yangchon-meon; d, Papyeong-myon; e, Munsan-eup; f, Baekhak-myon; g, Wangjing-meon; h, Misan-meon; i, Gimhwa-eup; j, Seo- myon; k, Cheorwon-eup; l, Geunnam-myon. 172 Korean J Parasitol Vol. 53, No. 2: 169-175, April 2015

and then increased thereafter once a blood collection program both years, followed by Cheorwon, Paju, and Gimpo (Table 1). had been initiated. The highest number of patients was report- Of the 425 inhabitants of Gimpo, 44 (10.4%) presented a ed in 2001 at 835 cases; however, only 90 cases were reported positive CSP-ELISA response. Haseong-myeon (Fig. 1A-a) pre- in the study areas (Fig. 2) (KCDC, personal communication). sented the highest infection rate (7/53, 13.2%) based on CSP- Of the 1,774 study subjects, 113 (6.4%) presented a positive ELISA, followed by Wolgot-myeon (Fig. 1A-b; 17/166, 10.2%) CSP-ELISA response. Gimpo (Fig. 1A) displayed the highest and Yangchon-myeon (Fig. 1A-c; 9.7%). Wolgot-myeon pre- positive CSP-ELISA rate (44/425, 10.4%), followed by Paju (Fig. sented the highest API in 2004 (1.42) and 2005 (0.71). 1B; 34/372, 9.1%), Yeoncheon (Fig. 1C; 19/451, 4.2%), and Haseong-myeon presented the second highest API in 2004 Cheorwon (Fig. 1D; 16/526, 3.0%). The API of 2004 was high- (0.69), but there were no malaria patients reported in 2005. er than that of 2005. The API was stable within each study area Yangchon-myeon had the third highest API in 2004 (0.50), from 2004 to 2005. Yeoncheon presented the highest API in but the second highest in 2005 (0.25) (Table 2). Of the 372 inhabitants of Paju, 34 (9.1%) presented a posi- 400 Gimpo Paju tive CSP-ELISA response. Papyeong-myeon (Fig. 1B-d) had a Yeoncheon 300 Cheorwon higher infection rate (25/140, 17.9%) than Munsan-eup (9/232, 3.9%) based on CSP-ELISA (Fig. 1B-e). Papyeong-my- 200 eon had a higher API than Munsan-eup in both 2004 and 2005. The correlations between the positive CSP-ELISA rates No. of patient 100 and API in 2004 (r =1.000, P =0.01) and 2005 (r =1.000, P =0.01) were significant (Table 3). 0 Of the 451 inhabitants of Yeoncheon, 19 (4.2%) presented 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 Year a positive CSP-ELISA response. Baekhak-myeon (Fig. 1C-f) presented the highest positive rate (13/265, 4.9%), followed Fig. 2. The number of malaria patients from 1993-2013 in the by Wangjing-myeon (Fig. 1C-g; 5/111, 4.5%) and Misan-my- study areas. ●, Gimpo-si; ■, Paju-si; ▲, Yeoncheon-gun; ▼, Cheorwon-gun. eon (Fig. 1C-h; 1/75, 1.3%), based on CSP-ELISA. Similarly,

Table 1. Positive rates of CSP-ELISA and annual parasite incidence

API Area No. of sera tested No. of positive sera Positive rate (%) 2004 2005 Gimpo-si 425 44 10.35 0.73 0.28 Paju-si 372 34 9.14 1.07 0.86 Yeoncheon-gun 451 19 4.21 2.99 1.99 Cheorwon-gun 526 16 3.04 1.13 1.20 Total 1,774 113 6.37 1.48 1.08

*Correlation coefficient between CSP-positive rate of 2004 and API of 2004 (r= -0.563, P = 0.437). *Correlation coefficient between CSP-positive rate of 2004 and API of 2005 (r= -0.799, P = 0.201). API, Annual parasite incidence.

Table 2. Positive rates of CSP-ELISA and annual parasite incidence in Gimpo-si

API Village No. of sera tested No. of positive sera Positive rate (%) 2004 2005 Haseong-myeon 53 7 13.20 0.69 0.00 Yangchon-myeon 206 20 9.71 0.50 0.25 Wolgot-myeon 166 17 10.24 1.42 0.71 Total 425 44 10.35 0.87 0.49

*Correlation coefficient between CSP-positive rate of 2004 and API of 2004 (r= -0.184, P = 0.882). *Correlation coefficient between CSP-positive rate of 2004 and API of 2005 (r= -0.672, P = 0.531). API, Annual parasite incidence. Lee et al.: Antibody-positive rate of P. vivax CSP and malaria incidence 173

Baekhak-myeon had the highest API in 2004 (3.69) and then Seo-myeon (0.49), and Cheorwon-eup (0.35) (Table 5). dropped to third place in 2005 (1.34). Misan-myeon had the second highest API in 2004 (2.79) but ranked first in 2005 DISCUSSION (2.79). Wangjing-myeon had the lowest API in 2004 (1.60) and ranked second in 2005 (2.40) (Table 4). The survey areas of this study are regions affected by a re- Of the 526 inhabitants of Cheorwon, 16 (3.0%) had a posi- emerging malarial outbreak in South Korea: Gimpo, Paju, tive CSP-ELISA response. Geunnam-myeon (Fig. 1D-l) had the Yeoncheon, and Cheorwon, which are located within 10-15 km highest infection rate (6/143, 4.2%), followed by Seo-myeon of the southern DMZ [8]. The DMZ is a 4-km-wide and (Fig. 1D-j; 4/126, 3.2%), Cheorwon-eup (Fig. 1D-k; 4/142, 250-km-long corridor that extends across the middle part of the 2.8%), and Gimhwa-eup (Fig. 1D-i; 2/115, 1.7%) based on Korean peninsula. No civilians have been allowed to enter the CSP-ELISA. Seo-myeon had the highest API in 2004 (1.98), DMZ for more than 50 years; therefore, natural landscape eco- followed by Gimhwa-eup (1.54), Cheorwon-eup (0.86), and systems and biodiversity are highly conserved in the DMZ [17]. Geunnam-myeon (0.45). However, Gimhwa-eup had the The outbreak areas are expanding annually, both in the south- highest API in 2005, followed by Geunnam-myeon (2.23), ern and eastern directions from the DMZ. The outbreak areas

Table 3. Positive rates of CSP-ELISA and annual parasite incidence in Paju-si

API Village No. of sera tested No. of positive sera Positive rate (%) 2004 2005 Munsan-eup 232 9 3.88 1.00 0.82 Papyeong-myeon 140 25 17.86 1.61 1.20 Total 372 34 9.14 1.31 1.01

*Correlation coefficient between CSP-positive rate of 2004 and API of 2004 (r= 1.000, P < 0.01). *Correlation coefficient between CSP-positive rate of 2004 and API of 2005 (r= 1.000, P < 0.01). API, Annual parasite incidence.

Table 4. Positive rates of CSP-ELISA and annual parasite incidence in Yeoncheon-gun

API Village No. of sera tested No. of positive sera Positive rate (%) 2004 2005 Wangjing-myeon 111 5 4.50 1.60 2.40 Baekhak-myeon 265 13 4.91 3.69 1.34 Misan-myeon 75 1 1.33 2.79 2.79 Total 451 19 4.21 2.69 2.18

*Correlation coefficient between CSP-positive rate of 2004 and API of 2004 (r= 0.025, P = 0.984). *Correlation coefficient between CSP-positive rate of 2004 and API of 2005 (r= -0.778, P = 0.433). API, Annual parasite incidence.

Table 5. Positive rates of CSP-ELISA and annual parasite incidence in Cheorwon-gun

API Village No. of sera tested No. of positive sera Positive rate (%) 2004 2005 Gimhwa-eup 115 2 1.74 1.54 2.46 Seo-myeon 126 4 3.17 1.98 0.49 Geunnam-myeon 143 6 4.20 0.45 2.23 Cheorwon-eup 142 4 2.82 0.86 0.35 Total 526 16 3.04 1.21 1.38

*Correlation coefficient between CSP-positive rate of 2004 and API of 2004 (r= -0.545, P = 0.455). *Correlation coefficient between CSP-positive rate of 2004 and API of 2005 (r= -0.090, P = 0.910). API, Annual parasite incidence. 174 Korean J Parasitol Vol. 53, No. 2: 169-175, April 2015

are believed to have originated from the northern part of the cubation-period patients who were infected in the relevant year DMZ. Since human passage through the DMZ is almost impos- and long incubation-period patients who were infected in the sible (although there are some exceptions in the Gaeseong In- previous year. Therefore, the positive anti-CSP antibody rates dustrial Zone), the reemergence of malaria in this area is pre- were compared with the APIs in the relevant and following sumed to have originated not from the immigration of infected years. In addition, sera obtained from inhabitants who lived in people from the north, but rather from mosquitoes infected non-epidemic areas were assayed to determine the false-posi- with P. vivax that flew from the north. The corridor is heavily tive rate of CSP antigen by western blotting. Three of 28 sam- fortified on both sides of the buffer zones with land mines and ples displayed a positive reaction (10.7%) in preliminary exper- barbed wire fences. Therefore, it is believed that these areas are iments. However, the possibility that these 3 positive cases had exposed to vector mosquitoes. To estimate the prevalence of never been in a malaria epidemic area in Korea could not be malaria exposure in these high-risk areas in Korea, the expres- ruled out. Therefore, it was concluded that whether malaria pa- sion of 2 recombinant proteins were evaluated. One is CSP, tients had travelled in high-risk areas in Korea should be con- which is a sporogony-stage protein that consists of the surface sidered. membrane common to all plasmodial sporozoites. CSP has a The incidence of malaria peaks in August after the rainy sea- central immunodominant region consisting of short tandem- son and declines to baseline by mid-October. Therefore, blood repeat amino acid sequences that contain multiple copies of collection was carried out between late-October and mid-De- the immunodominant B-cell epitope [9]. Because of its high cember, when the active anopheline population has dimin- immunogenicity and ability to induce a protective response in ished. Antibody detection might provide useful information sporozoite-immunized experimental animals and humans, concerning P. vivax infection in a previously naïve population. CSP is being investigated as a candidate molecule for the devel- Therefore, the correlations between the CSP-positive rate and opment of a human malaria vaccine. The immunodominant B- incidence of malaria patients were compared in high-risk ar- cell epitopes of CSP from a large number of isolates of P. falci- eas. The correlations between the positive CSP-ELISA rate and parum of diverse geographical origin and of a smaller number APIs of 2004 and 2005 were strongly negative, but the API of of isolates of P. vivax have been examined and were found to be 2005 showed a much stronger negative correlation with CSP conserved within each species [18]. positivity compared to that of 2004 in all 4 cities (Table 1). Interestingly, the maximum lifespan of the CSP antibody in The correlation between the positive rate of CSP-ELISA and human beings was determined to be 27 days in inhabitants of API of 2004 was slightly negative, but was strongly negative Thailand, and could not be boosted by additional exposure to with the API of 2005 in Gimpo-si (Table 2). The correlations CSP antigen, i.e., additional infection by anopheline mosqui- between the positive rates of CSP-ELISA and APIs of 2004 and toes [19]. These findings motivated us to consider the case of 2005 were strongly and significantly positive in Paju-si (Table long incubation-period patients, who usually present with on- 3). There was no correlation between the positive rate of CSP- set the year following infection after a 5-month-long winter ELISA and API of 2004 in Yeoncheon-gun, but a strongly nega- season without mosquitoes. Furthermore, the mean incubation tive correlation with the API of 2005 was observed (Table 4). period of P. vivax has been reported to be as long as 279±41 The correlation between the positive rate of CSP-ELISA and days (range, 153-452 days) [20]. This means that these long in- API of 2004 was strongly negative in Cheorwon-gun, but there cubation patients could display either the absence of or a re- was no correlation with the API of 2005 (Table 5). The differ- duced antibody level against CSP antigen. The percentages of ences in these results might be related to the different malaria cases with short or long incubation periods were 25% and control programs in these areas. Malaria control is carried out 75%, respectively [8]. However, it is possible that the CSP anti- by 4 different PHCs. Even with the main guidelines from the body level was elevated in short incubation-period patients KCDC, each PHC has their own manual that takes into ac- who presented with onset within a month after exposure to count local conditions of malaria prevalence and geographical CSP antigen via infective anopheline mosquitoes. Therefore, characteristics. To evaluate malaria transmission in a given the CSP antigen was selected among the many possible malaria geographical region, many factors, such as temperature, mos- antigens for this study. The hypothesis suggested by our malaria quito density, vector capacity, climate, rainfall, and humidity, team is that the API in a given year accounts for both short in- should be considered [21]. However, collecting such data re- Lee et al.: Antibody-positive rate of P. vivax CSP and malaria incidence 175

quires great effort. Therefore, it is necessary to establish a cost- 5. Soh CT, Lee KT, Im KI, Min DY, Ahn MH, Kim JJ, Yong TS. Cur- effective tool to analyze the current and future transmission rent status of malaria in Korea. Yonsei Rep Trop Med 1985; 16: 11-18. patterns of malaria in specific areas using serodiagnostic meth- 6. Chai IH, Lim GI, Yoon SN, Oh WI, Kim SJ, Chai JY. Occurrence ods. Parasitemia provides a classical means of measuring ma- of tertian malaria in a male patient who has never been abroad. laria endemicity, but patient incidence alone cannot provide a Korean J Parasitol 1994; 32: 195-200 (in Korean). complete understanding of malaria prevalence since many fac- 7. Cho SY, Kong Y, Park SM, Lee JS, Lim YA, Chae SL, Kho WG, Lee tors affect malaria prevalence in Korea, such as the population JS, Shim JC, Shin HK. Two vivax malaria cases detected in Korea. density of mosquitoes, vectorial capacity, long to short incuba- Korean J Parasitol 1994; 32: 281-284. 8. Lee JS, Kho WG, Lee HW, Seo M, Lee WJ. Current status of vivax tion period ratio, symptomatic to asymptomatic patient ratio, malaria among civilians in Korea. Korean J Parasitol 1998; 36: differences in rainfall and temperature, and immunity of the 241-248. community. The present study found that antibody detection 9. Arnot DE, Barnwell JW, Tam JP, Nussenzweig V, Nussenzweig methods were closely related to API values, which demon- RS, Enea V. Circumsporozoite protein of Plasmodium vivax: gene strates the value of this method for understanding malaria cloning and characterization of the immunodominant epitope. transmission in a given area even without knowledge of the Science 1985; 230: 815-818. 10. Lee HW, Lee JS, Lee WJ, Cho SH, Lee HS. The evaluation of re- aforementioned factors. combinant circumsporozoite protein in malaria diagnosis. Ko- In summary, antibody detection using CSP-ELISA may pro- rean J Microbiol 1999; 36: 142-149 (in Korean). vide useful information regarding malaria prevalence in cer- 11. Cerutti C Jr, Boulos M, Coutinho AF, Hatab Mdo C, Falqueto A, tain areas and individuals. These serological methods are use- Rezende HR, Duarte AM, Collins W, Malafronte RS. Epidemio- ful for identifying areas that require malaria control and for logic aspects of the malaria transmission cycle in an area of very low incidence in Brazil. Malar J 2007; 6: 33. evaluating the surveillance system in certain areas. 12. Collins WE, Skinner JC. The indirect fluorescent antibody test for malaria. Am J Top Med Hyg 1972; 21: 690-695. ACKNOWLEDGMENTS 13. Wang DQ, Tang LH, Gu ZC, Zheng X, Yang MN. Application of the indirect fluorescent antibody assay in the study of malaria We are grateful to all blood donors and the staff members infection in the Yangtze River Three Gorges Reservoir, China. of the Public Health Centers in Gimpo, Paju, Yeoncheon, and Malar J 2009; 8: 199. 14. Moody A. Rapid diagnostic tests for malaria parasites. Clin Mi- Cheorwon, Korea. This work was supported by the Korean Na- crobiol Rev 2002; 15: 66-78. tional Institute of Health and Inha University Research Fund 15. McKenzie FE, Prudhomme WA, Magill AJ, Forney JR, Permpan- (2014). ich B, Lucas C, Gasser RA Jr, Wongsrichanalai C. White blood cell counts and malaria. J Infect Dis 2005; 192: 323-330. CONFLICT OF INTEREST 16. Cohen J. Statistical power analysis for the behavioral sciences, 2nd ed. Psychology Press; 1988. 17. Kim KC. Preserving biodiversity in Korea’s demilitarized zone. We declare that we have no conflict of interest. Science 1997; 278: 242-243. 18. Shi YP, Alpers MP, Povoa MM, Lal AA. Diversity in the immuno- REFERENCES dominant determinants of the circumsporozoite protein of Plas- modium falciparum parasites from malaria endemic regions of 1. Mendis K, Sina BJ, Marchesini P, Carter R. The neglected burden Papua New Guinea and Brazil. Am J Trop Med Hyg 1992; 47: of Plasmodium vivax malaria. Am J Trop Med Hyg 2001; 64: 97- 844-851. 106. 19. Webster HK, Boudreau EF, Pang LW, Permpanich B, Sookto P, 2. Hasegawa Y. Malaria in Korea. Chosen Igakkai Zasshi 1913; 4: Wirtz RA. Development of immunity in natural Plasmodium fal- 53-69 (in Japanese). ciparum malaria: antibodies to the falciparum sporozoite vaccine 3. National Malaria Eradication Service, Ministry of Health and So- 1 antigen (R32tet32). J Clin Microbiol 1987; 25: 1002-1008. cial Affairs, Republic of Korea. Malaria pre-eradication pro- 20. Kho WG, Jang JY, Hong ST, Lee HW, Lee WJ, Lee JS. Border ma- gramme in Korea, 1961-1965. Progress Report. 1966, pp 44-70 laria characters of reemerging vivax malaria in the Republic of (in Korean). Korea. Korean J Parasitol 1999; 37: 71-76. 4. Paik YH, Rhee HI, Shim JC. Malaria in Korea. Jpn J Exp Med 21. Snow RW, Gilles HM. The Anopheles vector. In Essential Malari- 1988; 58: 55-66. ology. 4th ed. London, UK. Arnold. 2002, p 59-84.